Virulent phages to control Listeria monocytogenes in foodstuffs and in food processing plants

ABSTRACT

The present invention relates to virulent (lytic)  Listeria monocytogenes  phage from the Myoviridae family, preferably P100, alone or in combination with other virulent phages. P100 and the endolysin from P100 can be administered to food products, to the components that will be added to food products, and/or to the infrastructure of the food processing plants within which such food products are processed, or the containers or wraps in which such foods are stored and/or shipped, in order to reduce  Listeria monocytogenes  contamination. P100 can also be used in the present invention to identify  Listeria monocytogenes  bacteria present on (or within) foodstuffs, as well as those  Listeria monocytogenes  bacteria present in the equipment or the general environment of the food processing plants in which the foodstuffs are being processed and in animals infected with  Listeria monocytogenes . The phage and the endolysin of the present invention can also be used to treat animals infected with  Listeria monocytogenes . P100 will kill the bacteria that are within its host range with great efficiency and will propagate to high titer thereon. P100 can be combined with other lytic phage, and/or with other antimicrobial agents to reduce or eliminate  Listeria.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/US03/21061, filed Jul. 7, 2003, and designating the U.S., whichclaims priority from provisional applications 60/393,842 and 60/393,841,both filed Jul. 8, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of a particular class ofbacteriophages (“phage”) known as virulent phages that are lytic for thebacterial species Listeria monocytogenes, and which is shown in thepresent invention to reduce the counts of these bacteria and/or toprevent their growth in the first place, and to be useful foridentifying Listeria monocytogenes, in foods products (including but notlimited to the dairy industry) as well as on processing equipment andother sites in food industry facilities, and as a therapeutic agent fortreating animals infected with Listeria monocytogenes. One specificexample of a virulent Listeria monocytogenes phage, is a phagedesignated P100, recently discovered by one of the present inventors.The present invention also relates to an endolysin produced by P100 andthe use of the endolysin for reducing the amount of Listeriamonocytogenes in foods products as well as on processing equipment andother sites in food industry facilities, and in or on animals infectedwith Listeria monocytogenes and to methods that will enable additionalphage that have lytic properties to be developed and/or isolated.

Phages, as antibacterial agents, have the advantage of replicatingwithin the bacterial target. Thus, when their progeny lyse the cell andescape into the extracellular milieu, they can infect and multiply insucceeding generations of bacteria, producing progeny levels far greaterthan that of the binary growth of the target bacteria, therebyincreasing the phage population exponentially in numbers at the expenseof the bacterial targets.

The concept of using phages to identify bacterial contamination in foodproducts {and facilities, equipment}, in general, has been described inthe scientific literature (see, e.g. Greer, J. Food Prot., 49:104-109,1986). The concept of using specific Listeria phages in particular, toidentify Listeria contamination of dairy products andfacilities/equipment in specific, was described as early as 1990(Loessner et. al., Applied and Environmental Microbiology, Jun. 1990, p.1912-1918). The present invention concern the use of a recentlydiscovered Listeria phage with specific, essential and relevantproperties, which makes it particularly suitable for identifying andcontrolling Listeria contamination of dairy products, facilities andequipment.

In addition to the general scientific literature on the subject, thereis also patent literature that teaches the utility of phages in generalto control bacterial contaminations in food processing plants and infoodstuffs. See for example U.S. Pat. No. 5,006,347 issued on Apr. 9,1991, U.S. Pat. No. 4,851,240 issued on Jul. 25, 1989, GB 2 253 859 Apublished on Sep. 23, 1992 and EP 0414304A2 published on Feb. 27, 1991.However, none of the above discussed patents disclose a Listeria phagewhich was actually tested and shown to successfully control bacterialcontamination in food processing plants and in food products. The reasonfor this is that all of the Listeria phages known in the art at the timeof the disclosure in the previous patents were temperate phages, andwere therefore not efficient at nor suitable for industrial bacterialeradication purposes. The term “temperate” refers to the fact when astrain of phage injects its DNA into a bacterial target, the phage DNAintegrates into the DNA of the host cell, as a “prophage”, and canremain integrated therein for considerable periods of time. Since theprophage excises (and initiates replication and lysis) only when thehost cell becomes stressed, the ensuing bacterial lysis is unpredictableand not easily controlled, which is why temperate phages do not lendthemselves well to industrial applications. Temperate phages areunsuitable for industrial decontamination purposes for other reasons aswell, including the fact that they can deliver unwanted and dangerousgenes to the bacteria target into which their DNA integrates. Incontrast, there is a class of phages that lyse bacterial targetsdirectly, given that they do not have the molecular machinery requiredto integrate into the bacterial targets. Such phages are referred to asbeing “virulent” or “lytic” for the bacterial targets. Virulent phagesagainst Listeria monocytogenes were discovered recently, by one of thepresent inventors.

The first of these virulent Listeria phages, designated A511, wasdescribed in the literature in 1990 (see Loessner et al., Applied andEnvironmental Microbiology, Jun. 1990, p. 1912-1918, 1990). See also DE43 26617 C; Loessner et al., Applied and Environmental Microbiology,April 1996, vol. 62, No. 4, p. 1133-1140; and Gaeng et al., Applied andEnvironmental Microbiology, July 2000, vol. 66. No. 7, p. 2951-2958. Thevirulent phage according to the present invention belong to theMyovirdae family and have tails which contract towards the virus head.One particularly preferred phage is designated P100 and was deposited atthe American Type Culture Collection, 10801 University Blvd., ManassasVa. 20110-2209 on May 23, 2002, 2002, ATCC patent deposit designationnumber PTA-4383.

The virulent phages described in the present invention can also be usedagainst CFUs (colony forming units) of Listeria monocytogenes bacteriathat are in biofilms, as opposed to CFUs that are planktonic. The use oftemperate Listeria monocytogenes phages against Listeria biofilms hasbeen described in the literature (see e.g. Roy et. al., Appl. Environ.Microbiol., September, 59(9):2914-7, 1993). Specifically, Roy et. al.used temperate Listeria bacteriophages H387, H387-A, and 2671 of theSiphoviridae family. While these temperate phages demonstrated someefficacy in clearing a Listeria biofilm, even when used in combinationthe best they could obtain was a 3.5-3.7 log reduction in counts. Roy etal indicates that such reductions “will have to be improved on to meetthe recommended reduction level of 99.999% in a 30-s exposure for achemical sanitizing agent”. As stated above, temperate phages are notpredictable or readily controllable in the timing of or efficiency withwhich they can kill the target bacteria.

In addition to the use of the virulent phages described above, thepresent inventors have also found that virulent substrains can bederived from a number of temperate phage strains. These virulentsubstrains can be selected by techniques such as plaque isolation (inwhich one selects the clearest areas of a plaque, and enriches for themost virulent strains therein by repeated cycles of growing to hightiter, plating for new plaques, and picking the clearest areas of thelater-generation plaques). Examples of temperate phage strains, fromwhich virulent substrains have been or will be developed, include thetemperate strains designated A118, A502, A006, A500, PSA, P35, andrelated viruses.

The detection of Listeria is carried out in a known manner by means ofprocedures based on the culture of the microorganisms. The proceduredescribed in Int. J. Food Microbiol. 4 (1987), 249-256 takes two weeks.A somewhat more rapid procedure is recommended by the InternationalDairy Foundation (IDF); however it takes at least 6-8 days. Because oftheir length, both procedures are unsuitable for a rapid identification.Both procedures are moreover labor-intensive, as for the production ofindividual colonies nutrient media have to be inoculated several times,and as the isolates then have to be characterized by means ofbiochemical and serological investigation methods.

A prerequisite for the use of immunological tests is that the antigen isexpressed, which is not the case for all proteins at any time. The testsadmittedly last only a few hours, but in these procedures a two-daypre-enrichment culture is needed, as the detection limit is 10-1000×10³cells. DNA probes have a detection limit of the same order of magnitude.A prior multiplication of the bacteria is therefore also necessary forthese procedures: foodstuff samples or dilutions thereof are streakedout on agar plates, and the inoculated plates are incubated and theninvestigated in the colony hybridization procedure using aradio-labelled DNA probe. Detection is carried out by autoradiography.This method too is moreover labor and time-consuming.

The polymerase chain reaction (PCR) allows the in vitro replication ofnucleic acids; a preculture is in general not necessary in thisprocedure. The detection limit is 0.5×10³ cells. As, however, DNA fromdead cells or alternatively isolated DNA is also replicated in thisprocedure, contamination with dead cells cannot be differentiated fromcontamination with living cells.

The limitations of these methods indicate the need to provide improvedagents and methods for the detection of bacteria of the genus Listeria.

EP 0 168 933 (U.S. Pat. No. 4,861,709) discloses a detection procedurefor bacteria, e.g., Escherichia coli, based on the use of a recombinantbacteriophage. This phage contains the lux gene from Vibrio fischeri andthus makes possible the detection of E. coli by bioluminescence with agood detection limit (0.5×10³ cells). Using temperate phages, G. J.Sarkis et al. (1995) Molecular Microbiology 15, 1055-1067 describe adetection procedure for mycobacteria. In these detection procedures,only metabolically active bacterial cells are detected; interference dueto dead bacterial cells as in the PCR technique does not occur.

Scherer, et al., U.S. Pat. No. 5,824,468, issued on Oct. 20, 1998describes a detection procedure for bacteria of the genus Listeria,where a DNA vector is prepared which includes a genetic systemcomprising DNA which encodes the expression of one or more detectableproteins, and a DNA vector A511 is used which specifically infects thebacteria of the genus Listeria and transfers the genetic system to thebacteria. The detectable proteins are expressed in the bacteria anddetection of the detectable proteins indicates the presence of bacteriaof the genus Listeria.

Phage-encoded lysins or endolysins are highly active enzymes whichhydrolyze bacterial cell walls. These phage encoded cell wall lyticenzymes are synthesized late during virus multiplication and mediate therelease of progeny virions. Endolysins can be used to lyse Listeriacells to recover nucleic acids or cellular protein for detection ordifferentiation. The endolysin can also be used to treat animals(including humans) which are infected with Listeria and to treatsurfaces which may be contaminated with Listeria. Lysins from Listeriaphages (A118, A500 and A511) have been cloned and purified (Loessner, etal., Applied and Environmental Microbiology, August 1996, p. 3057-3060).

2. Description of the Related Art

Listeria monocytogenes is a bacterial pathogen that contaminates manyfood products, the list of which includes but is not limited to softcheeses, patés, ice cream, smoked and cured fish, frozen seafood,salads, and processed meats. When ingested, these bacteria can produce adisease termed listeriosis, characterized by a variety of symptoms andconditions, including diarrhea, abortion, and encephalitis.Collectively, in the industrialized nations, hundreds of deaths occureach year as a result of Listeria monocytogenes food contamination.

The food processing industry has not been sufficiently successful ineradicating Listeria monocytogenes bacteria from the environment of theprocessing plants. As a result, even foods that have been pasteurized attemperatures high enough to kill these bacteria nevertheless becomecontaminated, post-pasteurization. The bacteria gain access to thefoodstuffs through one or more routes, including (i) from the rawmaterials (e.g. raw milk, and/or milk that has been pasteurized at lowtemperatures); (ii) from the processing machinery (in and on which thebacteria can grow as biofilms that are difficult to eradicate ); and(iii) from airborne bacteria present in the plant environment which cansettle onto the surface of the foodstuffs during curing, packaging, andso on.

Despite the numerous methods used in the food industry to control andprevent L. monocytogenes contamination, the bacteria gain access to andpersist in the environment of food processing plants. Moreover, theysurvive the very high concentrations of salt that are present in severalfood-making processes. The resulting contamination of the foodstuffs(including but not limited to cheeses, pates, cold cuts, hot dogs andother processed foods) leads to scores of deaths each year in developednations, and also to product recalls whose retail worth each year, inthe aggregate, is measured in the hundreds of millions of dollars.

The methods currently in use to control Listeria in the food industryinclude: (i) pasteurization of primary ingredients (e.g. milk) and heattreatment of the products, which is often unsuccessful becauserecontamination frequently occurs and many products cannot undergo afinal (listeriocidal) heat treatment; (ii) application ofphysicochemical agents such as disinfectants, enzymes, antibiotics,etc., which experience has shown do not reduce the bacterial countssufficiently; and (iii) attempts to break up biofilms mechanically,which leave sufficient residues of bacteria behind that the foodstuffsstill become contaminated.

Additional methods must therefore be made available to the foodprocessing industry in order to protect the health of consumers, and toreduce the exposure of numerous companies to the great cost and the lossof good will that result from such contaminations and recalls.

The present inventors have conducted a series of experiments using astrain of L. monocytogenes that is prevalent in the processing plant ofa particular manufacturer of soft (“spread”) cheeses. That bacterialstrain proved to be susceptible to phage P100 in vitro. As will be shownin the Examples section, phage P100 proved able to reduce belowmeasurable/detectable limits the Listeria bacteria that had been spikedinto a cheese-like matrix.

SUMMARY OF THE INVENTION

Virulent phage P100, as well as other virulent phages from theMyoviridae and Siphoviridae families, and virulent mutants of varioustemperate strains of phage (such as but not limited to phages B054,A118, A502, A006, A500, PSA, P35 and related viruses) are used in thepresent invention to control Listeria monocytogenes bacteria present on(or within) foodstuffs, as well as those Listeria monocytogenes bacteriapresent in the equipment or the general environment of the foodprocessing plants in which the foodstuffs are being processed. Thesephage can also be used to treat animals infected with Listeriamonocytogenes.

Virulent phage P100 is used in the present invention to identifyListeria monocytogenes bacteria present on (or within) foodstuffs, aswell as those Listeria monocytogenes bacteria present in the equipmentor the general environment of the food processing plants in which thefoodstuffs are being processed and in animals infected with Listeriamonocytogenes. In the present invention, a recombinant DNA vector isprepared using virulent phage P100 which is specific for Listeriamonocytogenes. The vector includes a genetic system comprising DNA whichencodes the expression of one or more detectable proteins which are nota gene product of Listeria monocytogenes. The DNA vector infects thebacteria of the genus Listeria and transfers the genetic system to thebacteria. The detectable proteins are expressed by the bacteria and thedetection of the detectable proteins indicates the presence of bacteriaof the genus Listeria.

An endolysin derived from P100 is used to reduce the counts of Listeriamonocytogenes and/or to prevent their growth in the first place, infoods products (including but not limited to the dairy industry) as wellas on processing equipment and other sites in food industry facilities,and as a therapeutic agent for treating animals infected with Listeriamonocytogenes. Endolysins from Listeria phages have high substratespecificity and almost exclusively lyse Listeria cells.

The present invention is directed to the use of a class of Listeriamonocytogenes phages which are particularly suitable for bacterialcontrol methods and for the detection of Listeria monocytogenes. Thephage are preferably from the Myoviridae family and are virulent againstListeria monocytogenes strains of serovar 1/2. In addition, the presentinvention isolates virulent mutants of temperate strains and uses thosespecific mutants in the control of bacterial contamination of foodstuffsand of food processing plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that when Listeria monocytogenes is inoculated at 10³ CFUper ml of liquid culture and phages added at a concentration of 5×10⁸PFU per ml—virtually complete eradication of the Listeria bacteriaoccurs.

FIG. 2 shows that applying phage P100 to the surface of asurface-ripened cheese completely prevented outgrowth of Listeriamonocytogenes that had been spiked into the starter culture.

FIG. 3 shows phage titers on cheese (PFU/cm²).

DESCRIPTION OF PREFERRED EMBODIMENTS

By “vector” is meant a nucleic acid molecule that is capable ofself-replication when introduced into a suitable host cell. In general,the vectors used as starting materials for the recombinant vectors ofthe present invention are bacteriophages which are highly specific, andpreferably absolutely specific, for infecting bacteria of the genusListeria, and wherein the recombinant vectors retain that specificity.For example, a suitable vector is the Listeria bacteriophage P100, whichspecifically lyses bacteria of the genus Listeria (inevitably lytic). Itis a myovirus of complex construction. With respect to essentialfeatures, the Listeria phage P100 differs from many known Listeriaphages; the differences relate to morphology, host range, proteinprofiles (electrophoresis in SDS gel, isoelectric focusing, amino acidcomposition of the main structural proteins, DNA/DNA hybridization).

For detection of the presence of bacteria of the genus Listeria, markergenes are employed. These are genes which can be detected upon infectionby the vector of a suitable host cell and subsequent culturing of thecells under conditions suitable for expression of the marker genes. Itis preferred that the marker genes are those which do not occur in thebacteria of the genus Listeria, and which are inserted into the vector,the phage P100, using recombinant techniques. Such genes and their geneproducts are known in the art; they include bioluminescent proteins suchas the lux gene which occurs in variants in various luminescentbacteria, for example of the genus Vibrio. The incorporation of the luxgene allows detection by luminescence measurement. An example of the luxgene is gene luxAB from Vibrio harveyi. Other suitable proteins includebut are not limited to luciferase and fluorescent proteins such as greenfluorescent protein.

The detection reaction can take place on a solid surface including butnot limited to a test strip. In this embodiment, the vector containingthe marker gene could be reversibly immobilized in or downstream from asample application zone. Alternatively, the vector could be incubatedwith the sample prior to application on the test strip. Anti-listeriaantibodies would be irreversibly immobilized downstream from the vectorand the sample application zone. If a sample is applied which containsListeria, the vector would infect the Listeria and the detectableproteins would be expressed. As the sample moves down the test strip,the Listeria would become immobilized by the anti-listeria antibodies.The marker proteins would then be detected in the immobilized Listeria.

The endolysin of the present invention can be isolated by techniquesknown in the art including but not limited to lysis, chromatography,filtration, and centrifugation. The endolysin can be isolated fromListeria which have been incubated with P100 or the endolysin can becloned and expressed in a host bacteria (e.g. E. coli, L. lactis, S.aureus, and B. cereus). The endolysin can be isolated from the hostbacteria or the host bacteria containing the endolysin can be directlyapplied or administered without isolation of the endolysin. For example,a host bacteria which produces the endolysin could be administered to ananimal or applied to a surface where the endolysin would be secretedinto the food, onto the surface or into the animal's gut. The endolysincan then attack Listeria cells present in this environment. One unit ofendolysin activity is defined as the amount of endolysin necessary todecrease the optical density at 600 nm by 0.01/min, at pH 8.0 and 25° C.in a volume of 1 ml, when heat-killed, washed cells of Listeriamonocytogenes are used as a substrate.

The above-referenced endolysin, host bacteria containing the endolysinand/or phage is applied on or into food products, and/or into variousphysical sites within the food processing plants, by a number of meansincluding, but not limited to, admixing the endolysin, host bacteriacontaining the endolysin and/or phage into the food products, sprayingthe endolysin, host bacteria containing the endolysin and/or phage ontothe foodstuffs, spraying the endolysin, host bacteria containing theendolysin and/or phage onto the plant equipment, and/or directlyapplying the endolysin, host bacteria containing the endolysin and/orphage to the plant equipment. Said applications significantly reduce thenumbers of Listeria monocytogenes bacteria that would otherwise bepresent.

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can also be used to treat animals, includinghumans, infected with Listeria monocytogenes. Any suitable route ofadministration can be used to administer the phage including but notlimited to: oral, aerosol or other device for delivery to the lungs,nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal,vaginal, rectal, topical, lumbar puncture, intrathecal, and directapplication to the brain and/or meninges. Excipients which can be usedas a vehicle for the delivery of the phage, endolysin and/or hostbacteria containing the endolysin will be apparent to those skilled inthe art. For example, the free phage, endolysin and/or host bacteriacontaining the endolysin could be in lyophilized form and be dissolvedjust prior to administration by IV injection. The dosage ofadministration for the phage is contemplated to be in the range of about10³ to about 10¹³ pfu/per kg/per day, and preferably about 10¹² pfu/perkg/per day. The dosage of administration for the endolysin iscontemplated to be in the range of about 2-2000 ng/per g/per day, andpreferably about 20-200 ng/per g/per day. The phage, endolysin and/orhost bacteria containing the endolysin are administered until successfulelimination of the Listeria monocytogenes is achieved or until theamount of Listeria monocytogenes is substantially reduced.

The present invention also covers the use of the phages, endolysinand/or host bacteria containing the endolysin, when used in combinationwith other anti-Listerial agents known in the art. Examples of suchanti-Listerial agents, which are preferentially combined with phages,endolysin and/or host bacteria containing the endolysin, include but arenot limited to:

1. Endolysins (Phage Lysins):

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be combined with listeriolysins which areenzymes which have been shown to selectively control Listeria in foodand the enviromnnent (DE4326617C1 and EP 95932002.9)

2. Surface Disinfectants:

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be combined with known surface disinfectantssuch as (i) preservatives of various kinds, such as but not limited tobenzoic acid and BHT; and (ii) various disinfectants with which thephages are compatible, such as but not limited to quaternary ammoniumcompounds.

3. Antibiotics

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be used in combination with knownantimicrobial agents (including antibiotics and chemotherapeutic agents)including but not limited to vancomycin, nisin, danofloxacin andneomycin.

4. Enzymes

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be used in combination with enzymes to aid inbreaking up biofilms (e.g. biofilms found in food processing equipment).Such enzymes are known in the art and include but are not limited topolysaccharide depolymerase enzymes, and protease.

5. Surfactants

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be combined with known surfactants when usedto treat food processing equipment. The surfactant helps to wet thesurface so that the phage are properly distributed over the varioussurfaces, and to solubilize and remove dirt so that the Listeria areaccessible to the phage. Suitable surfactants include but are notlimited to Tween 80, 20 and 81 and Dobanols.

6. Bacteriophages Specific for Bacterial Contaminants Other thanListeria monocytogenes

The phage, endolysin and/or host bacteria containing the endolysin ofthe present invention can be combined with phage specific for Listeriamonocytogenes and/or phage specific for other bacteria known tocontaminate food processing equipment and food products. Such bacteriainclude but are not limited to E. coli, and bacterial species from thegenera Salmonella, Bacillus, Staphylococcus, Streptococcus, Clostridium,and Pseudomonas.

The phage can be applied in a liquid or a powdered form to food productsand food processing equipment. If applied as a liquid, the phage areapplied at a concentration of 10³ to 10¹⁰ PFU (plaque forming units) permL and preferably at a concentration of 10⁶ to 10⁹ PFU (plaque formingunits) per mL. If applied as a dry powder the phage are applied at aconcentration of 10³ to 10¹⁰ PFU (plaque forming units) per mg andpreferably at a concentration of 10⁶ to 10⁹ PFU (plaque forming units)per mg. The phage can be suspended in a suitable carrier prior toapplication or drying, including but not limited to protein solutionscontaining BSA, casein, whey protein, soy bean protein, etc and sugarbased carriers containing sugars such as mannitol. The phage can belyophilized or cryopreserved by vitrification and either suspended in asolution prior to application or applied directly as a dry powder.

Suitable amounts of phage for use in the present invention can beobtained by techniques known in the art, including but not limited to abatch technique where a culture of host bacteria is grown and thenseeded with phage. After an amount of time suitable to allow maximalphage propagation and bacterial lysis, the culture is further lysed byphysical or chemical means and the lysate spun down. The phagecontaining supernatant can be used as is or further purified usingtechniques such as ultrafiltration, chromatography and centrifugation.

The endolysin can be applied in a liquid or a powdered form to foodproducts and food processing equipment. The endolysin is applied in aconcentration between 2 to 2000 ng endolysin per ml or per gram ofcarrier, and preferably between 20 to 200 ng endolysin per ml or pergram of carrier.

As used in the present application, the term “dairy product” is intendedto include any food product made using milk or milk products, includingbut not limited to milk, yogurt, ice cream, cheese, butter, and cream.

As used in the present application, the term “meat product” is intendedto include any food product which contains animal tissue, including butnot limited to beef, pork, and poultry. The term “ready to eat meatproduct” in intended to include any meat product which does not requirecooking prior to consumption, including but not limited to patés, hotdogs, bologna, salami, and cold cuts.

As used in the present application, the term “fish product” is intendedto include any food product which contains tissue from an aquatic animalincluding but not limited to lobster, crab, fresh water and saltwaterfish and other seafoods.

As used in the present application, the term “unpasteurized foodproduct” is intended to include any food product which is prepared usingunpasteurized primary ingredients and which does not undergo a final(listeriocidal) heat treatment.

As used in the present invention, the term “salad” is intended toinclude any food product which contains mixtures of vegetables orfruits, and particularly such mixtures as are presented for consumers tochoose from in a display commonly referred to as a “salad bar”.

EXAMPLE 1

Eradication of Listeria monocytogenes in a Liquid Culture:

-   Step 1. 10³ CFUs of Listeria monocytogenes are mixed in a liquid    culture.-   Step 2. 5×10⁸ PFU of phage P100 are mixed into the liquid culture.-   Step 3. As a control, the buffer in which phage P100 was suspended    is mixed into an aliquot of the liquid culture.-   Step 4. Colony counts of the bacteria are performed at various    intervals of time.    Results are shown in FIG. 1.

EXAMPLE 2

A challenge experiment was done using a strain of anti-Listeriamonocytogenes phage known as P100, in a cheese model on lab scale. Thisexperiment incorporated technical flora to achieve the surface-ripeningqualities (taste, texture, etc.) characteristic of an establishedcommercial cheese-making process. The strain of Listeria monocytogenes(“Lm”) known as strain C that was used in this experiment is a commoncontaminant of a certain cheese-making plant.

Materials and Methods

Challenge Experiment on Cheese

-   -   The experiment was carried out on cheese taken directly from the        brine (unadjusted pH).    -   Lm strain C, technical flora, and phage P100 were applied on the        cheese at t=0 by plating 210 μL or 1 mL of incubation mix on 64        cm² cheese surface. t=0 is the same as “CMD+1” (Cheese Making        Day+1). Cheeses treated with 1 mL solution were subsequently        dried in a laminar flow cabinet to dry the surface. The        incubation mix consisted of:    -   110 g/L NaCl    -   Technical flora:    -   Debaryomyces hansenii NIZO F937 and NIZO F1200 (yeasts) at 10⁸        CFU/mL    -   Brevibacterium linens NIZO B1204 (a bacterium typical for red        smear cheese) at 10⁸ CFU/mL    -   LmC corresponding to a concentration of 7 cfu/cm² (diluted in        pfz from an exponential growing culture)    -   Phage P100 (in MPOS buffer) at a concentration corresponding to        1×10⁷ pfu/cm² or 5×10⁵ PFU/cm²    -   See Table 1 for exact treatment combinations    -   The cheeses were incubated at 14° C. and 98%-99% relative        humidity    -   The cheeses were treated daily with 210 μL or 1000 μL washing        solution that contains P100, at CMD+6, CMD+10, and CMD+13 and 1        mL/70 cm² (treatment combination 4 and 5)    -   The washing solution contained:    -   110 g/L NaCl    -   Brevibacterium linens NIZO B1204 at 10⁸ CFU/mL    -   Phage P100 (in MOPS buffer) at a concentration corresponding to        1 10⁷ PFU/cm² or 5 10⁵ PFU/cm²    -   Cheeses were packed using parchment paper, a material that is        used to pack Munster cheese, at CMD+16. (CMD+16 is packaging day        (PD))    -   Cheeses were stored at 6° C. until PD+63    -   LmC counts were analyzed at the time points indicated in        Table 1. Analysis was done before treatment with phage.    -   Quantitatively: samples of 70 cm² were cut out of the cheese and        analyzed on selective media.    -   Qualitatively at CMD+1 and CMD+37 by enrichment procedure.    -   Qualitative/Quantitative analysis will depend also on the level        of outgrowth of Listeria on the cheeses. If cell counts are        >10², no enrichments were done. If cell counts were lower,        enrichments were done also before packaging.    -   Furthermore analysis of pH and technical flora.    -   In one cheese at CMD+6, phage titers were determined before and        after application of phage.

Results

The ripening of the cheese was good, since yeast and Brevibacterium grewout well on the cheese and the cheese surface was de-acidified.

Listeria monocytogenes C grew well on the cheese surface in this model,to levels of 10⁵-10⁷ CFU/cm² in the negative controls (negative control:no phage applied) (FIG. 2).

As seen in FIG. 2, phage P100 completely inhibited growth of LmC. NoListeria was detected using the quantitative plate counting method, oron enrichment. The detection limit using the enrichment procedure is inthe order of magnitude of one Listeria per 60 cm². This indicates thatphage P100 not only inhibited growth but actually reduced Listeriatiters. As shown in FIG. 2 applying phage P100 to the surface of asurface-ripened cheese completely prevented outgrowth of Listeriamonocytogenes that had been spiked into the starter culture.

-   Key: CMD=Cheese-making day    -   PD=Packaging day

In order to determine if phages can survive on the cheese surface, inanother arm of the experiment (in which a high or a low dose of phagewas applied to the cheese surface), phage titers were determined atCMD+6, before as well as after application of phage. In all samples,phages had been applied at CMD+1, CMD+2, CMD+3, and CMD+4. Therefore, inthe samples taken before the application of phage (at CMD+6), phages hadbeen present on the cheese for at least 48 hours. Active phages wererecovered from the cheese surface at both doses (FIG. 3). The phagetiters of the samples taken before application of phage at CMD+6 werelower than the samples after application of phage. The increase in phagetiter corresponded well with the expected increase based on the doseadded (i.e., either 1×10⁷ pfu/cm² or 5×10⁵ PFU/cm²). The results showthat phage P100 remains active on the cheese surface for at leastseveral days.

EXAMPLE 3

Assay, Overexpression and Purification of Endolysins from E. coliJM109(pHPLxxx) Plate Assay

-   1. Prepare Listeria monocytogenes assay strain for activity testing:    grow 1000 ml overnight-culture in tryptose broth or trypticase soy    broth, centrifuge, wash cells once with SM Buffer, pH 8.0 (see    Sambrook et al., 1989), (or PBS), and resuspend in 20 ml SM Buffer    (or PBS) (approx. 50-fold concentration).-   2. Store in 1 ml amounts at −20° C. or at −80° C.-   3. Streak or plate E. coli JM109(pHPLxxx) on LB-agar (containing 100    μg/ml ampicillin), and incubate overnight.-   4. When colonies are of sufficient size, prepare replica plate (with    NC-filters) on LB-Amp plates supplemented with 1 mM IPTG. Incubate    for 5-7 hours until small colonies are visible.-   5. Expose surface of plate (upside down) to filter paper saturated    with chloroform, for 5 min.-   6. Quickly overlay the colonies with 3-4 ml of molten soft agar    (0.4%, in SM Buffer, approx. 45° C.), supplemented with 0.5 ml of a    50-fold concentrated L. monocytogenes culture (overlay should be    thin, even, and quite turbid).-   7. Incubate at RT for 60 min (up to overnight), until clear lysis    zones around colonies are visible. This can take any time, from 5    minutes to 5 hours. (this is an important assay; it must work.    Otherwise there might be a problem with the strain, or the activity    testing procedure.)    Production-   8. Prepare overnight culture of JM109(pHPLxxx) in LB broth with 100    μg/ml Amp @ 30-35° C. incubation.-   9. In the morning, inoculate 250 ml prewarmed broth with 10 ml O/N    culture, grow to OD600 of 0.5-0.6.-   10. Induction with 1 mM IPTG, incubate for further 3-4 h until the    growth rate begins to cease.-   11. Harvest cells by centrifugation, resuspend pellet(s) in 5 ml per    250 ml culture PBS (pH 8.0) 0.05% Tween20 or, if downstream Ni—NTA    purification is required, Buffer A (see below). Freeze cells at −20°    C.-   12. Thaw cells. Prepare cell extracts by French-Press;    centrifugation (>30000×g, 30 min); and filtration of supernatant    (0.2 μm filter, preferably made from PES). Store the enzyme    containing extract on ice (a few hours), or at −20° C. (note:    sonication can also be used, but yields obtained by French-Press are    generally much better).    Photometric Activity Assay (OD 600)-   13. Use freshly grown, mid-to-end-of-log-phase-cells, or the    previously frozen L. monocytogenes cells from Step 1 (above).    Resuspend in PBS Buffer, pH 8.0, 0.05% Triton X100 (adjust OD to    approx. 1.0-1.5). Use half-micro plastic cuvettes (1 ml); add 900 μl    cells, prewarm to at least RT but preferably 30-37° C., and add    50-100 ml enzyme. Turbidity should drop to 0.5 or below within 5    minutes or less.

This crude extract can be used for lysis of Listeria cells and releaseof chromosomal DNA, Plasmids, proteins and enzymes. However, it containshigh amounts of E. coli proteins, nucleic acid (DNA, RNA) fragments,ATP, and contaminating pHPL vector. If this is not desirable, purify theenzymes by IMAC (see below).

Purification

-   14. Fractionation of crude extracts with Ni—NTA-Resin. The procedure    outlined in steps 8-11 is for liquid chromatography (FPLC or    similar, see step 15), and is highly recommended. However, for small    scale purification, it can also be performed in a more simple    batch-type procedure:. Use approximately 3 ml resin (NiNTA Agarose)    per 250 ml initial culture volume. After exposure of resin to    proteins, carry out wash steps (in batches) by low speed    centrifugation (less than 500×g). After the last wash, harvest resin    and aliquot in portions of 1-2 ml into small disposable plastic    columns (available from BioRad and others); place into 15 ml conical    or round bottom tubes. Add elution buffer (approx 1 ml) (100% B),    let stand for 5 minutes, spin and collect liquid. Repeat elution 1-2    times. Continue with step 19.-   15. FPLC Purification: Prepare sufficient amounts of Buffer A (50 mM    phosphate buffer, pH 8.0, 500 Mm NaCl, 5 mM imidazole) and Buffer B    (same as A, but 250 mM imidazole). Use imidazole gradient with    Buffers A and B. Load extract (up to 40 ml=extract from 2 liters    (8×250 ml) of culture) on Ni—NTA column (25 ml volume), Use a low    flow rate (0.75 to 1.0 ml/min). If necessary, regenerate Ni—NTA    resin (Ni—NTA Superflow) before purification of each individual    enzyme by the procedure outlined in the handbook from Qiagen. A new    or regenerated column can be used for at least 5 runs.-   16. Wash with at 5 column volumes of 100% Buffer A (flow rate 2 to 3    ml/min)-   17. Wash with 3-5 column volumes of 12% Buffer B (total conc.    approx. 35 mM Imidazole), until the baseline reading (@ 280 nm) is    stable. This step will remove most contaminating proteins (be aware    that Imidazole itself increases the reading at 280 nm !).-   18. Elute enzyme fraction with 250 mM Imidazole (100% buffer B). It    is best to collect the peak manually (regarding the right shoulder    of the peak, stop early—don't be fooled by the higher absorption of    imidazole itself). Store on ice. Do not freeze the freshly eluted    fractions, the enzymes have a tendency to cold precipitate in the    presence of high salt and imidazole!-   19. Check all eluted fractions by photometric activity assay (see    above).-   20. Concentration and buffer-exchange (removal of imidazole and high    salt) of active fractions with centrifuge filter units (PES    membrane, Mr cut-off 10 kDa). Pre-treat membrane with Lysis Buffer    (PBS, pH 8.0, 0.05% Triton X100, 0.05% Tween20). Wash twice with 5    ml lysis buffer. In case membrane gets plugged with protein,    transfer to new filter unit. Filter—sterilize using a 0.2 μm syringe    filter (use PES membrane—Cellulose will retain much of the    enzymes!). Alternatively, buffer exchange can be performed by    dialysis of concentrated protein solution (PBS or Tris buffers,    0.05% Tween20). We found that HPL118 (but not HPL511 or HPL500) has    a tendency to aggregate during prolonged dialysis.-   21. Check fractions by SDS-PAGE, estimate protein purity (Should be    more than 90% pure), and protein concentration (typically in the    range of 2-4 mg/ml). If the preparation is not of sufficient purity,    try to wash column (Step 10) with 15% Buffer B, and elute HPL    proteins with 150 mM Imidazole (60% Buffer B), or 200 mM Imidazole    (80% Buffer B). One can also “reload” the entire initial protein    prep (dilute at least 10-fold before reloading, or dialyse to remove    imidazole) onto the column, and perform a 2nd step purification    using altered conditions, such as a pH gradient. Gel-filtration also    further increases protein purity. However, this is certainly not    needed for all standard applications.-   22. Adjust concentrated enzyme solution to a final content of 30-50%    Glycerol, aliquot into 50-500 μl portions, store at −25° C. Under    these conditions, enzymes are stable for several months. Freezing at    −80° C. sometimes resulted in loss of activity.

DISCUSSION AND CONCLUSION

When Listeria monocytogenes bacteria were spiked onto the surface of asurface-ripened cheese along with the starter culture, applications of asufficient dosage of Phage P100 to the surface completely eradicated thebacteria, as confirmed by enrichment studies (detection limit usingenrichment: one Listeria CFU per 60 cm² of cheese surface). In allcheeses treated with phage (high or low concentration, one applicationor multiple), Listeria titers after treatment with P100 were lower thanin the controls. Listeria emerged only when phages were applied onlyonce (instead of on multiple time points) or at low concentration.Phages can remain active on cheese for several days.

1. A method for controlling Listeria contamination of a food product,food processing equipment, or a food storage container, comprisingapplying lytic phage P100, ATCC patent Deposit Accession No. PTA-4383,to said food product, equipment or container in an amount sufficient toreduce the amount of Listeria, thereby controlling said Listeriacontamination.
 2. The method according to claim 1, wherein said P100 isapplied in combination with phage A511, ATCC Patent Deposit AccessionNo. PTA-4608.
 3. The method according to claim 1, wherein said lyticP100 phage is applied in combination with at least one additional agentselected from the group consisting of listeriolysin, a surfacedisinfectant, an antibiotic, a surfactant, an enzyme, and a phage thatlyses a contaminating bacteria other than Listeria monocytogenes.
 4. Themethod according to claim 1, wherein said food product is a dairyproduct.
 5. The method according to claim 1, wherein said food productis an unpasteurized food product.
 6. The method according to claim 1,wherein said food product is a meat product.
 7. The method according toclaim 6, wherein said meat product is a ready to eat meat product. 8.The method according to claim 1, wherein said food product is a fishproduct.
 9. The method according to claim 1, wherein said container is asalad bar and said food product is salad.
 10. The method according toclaim 1, wherein said food processing equipment is selected from thegroup consisting of (i) tube through which milk is being pumped, (ii) ahigh-salt content tank for processing cheese, (iii) a container fromwhich cultures are applied to a surface of a cheese, (iv) a set ofshelves on which a product is dried and cured, and (v) a floor drain.11. The method according to claim 1, wherein said lytic P100 phage isapplied by mixing the phage with a liquid or semi-solid food product.12. The method according to claim 1, wherein said lytic P100 phage issuspended in a liquid and sprayed onto a surface of said food product,equipment or container.
 13. The method according to claim 12 whereinsaid lytic P100 phage are sprayed onto said food processing equipmentsurface in combination with an agent selected from the group consistingof listeriolysin, a surface disinfectant, an antibiotic, a surfactant,an enzyme, and a phage that lyses contaminating bacteria other thanListeria monocytogenes.
 14. The method according to claim 1, whereinsaid lytic P100 phage is lyophilized or cryopreserved by vitrificationand applied in a dry form to said food product, equipment or container.15. A composition comprising an isolated P100 phage, ATCC Patent DepositAccession Number PTA-4383 in a carrier.
 16. The composition according toclaim 15, further comprising, in said carrier, an isolated A511 phage,ATCC Patent Deposit Accession Number PTA-4608.
 17. The compositionaccording to claim 15, farther comprising an agent selected from thegroup consisting of listeriolysin, a surface disinfectant, anantibiotic, a surfactant, an enzyme, and a phage that lysescontaminating bacteria other than Listeria monocytogenes.
 18. Thecomposition according to claim 15, wherein said carrier is apharmaceutically acceptable carrier.
 19. An isolated P100 phage asdeposited at the American Type Culture Collection, ATCC Patent DepositAccession Number PTA-4383.